Most bacterial infections can be treated with antibiotics such as penicillin, discovered decades ago. However, such drugs are useless against viral infections, including influenza, the common cold, and deadly hemorrhagic fevers such as Ebola. [*/quote*]

Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, Massachusetts, United States of AmericaAbstract Top

Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.

Funding: This work is funded by grant AI057159 (http://www.niaid.nih.gov/Pages/default.a​spx) from the National Institute of Allergy and Infectious Diseases and the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, with previous funding from the Defense Advanced Research Projects Agency, Defense Threat Reduction Agency, and Director of Defense Research & Engineering. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the United States government.

A serious threat is posed by viral pathogens, including clinical viruses (HIV, hepatitis viruses, etc.), natural emerging viruses (avian and swine influenza strains, SARS, etc.), and viruses relevant to potential bioterrorism (Ebola, smallpox, etc.). Unfortunately, there are relatively few prophylactics or therapeutics for these viruses, and most which do exist can be divided into three broad categories [1]–[3]: (1) Specific inhibitors of a virus-associated target (e.g., HIV protease inhibitors, RNAi) generally must be developed for each virus or viral strain, are prone to resistance if a virus mutates the drug target, are not immediately available for emerging or engineered viral threats, and can have unforeseen adverse effects. (2) Vaccines also require a new vaccine to be developed for each virus or viral strain, must be administered before or in some cases soon after exposure to be effective, are not immediately available for emerging or engineered viral threats, can have unforeseen adverse effects, and are difficult to produce for certain pathogens (e.g., HIV). (3) Interferons and other pro- or anti-inflammatories are less virus-specific, but still are only useful against certain viruses, and they can have serious adverse effects through their interactions with the immune and endocrine systems.

To overcome these shortcomings of existing approaches, we have developed and demonstrated a novel antiviral approach that is effective against a very broad spectrum of viruses, nontoxic in vitro and in vivo, and potentially suitable for either prophylactic or therapeutic administration. Our approach, which we call a Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO), is designed to selectively and rapidly kill virus-infected cells while not harming uninfected cells.

The second natural process used by our approach is one of the last steps in the apoptosis pathway [14], in which complexes containing intracellular apoptosis signaling molecules, such as apoptotic protease activating factor 1 (Apaf-1) [15]–[16] or FLICE-activated death domain (FADD) [17]–[18], simultaneously bind multiple procaspases. The procaspases transactivate via cleavage, activate additional caspases in the cascade, and cleave a variety of cellular proteins [14], thereby killing the cell.

Many viruses attempt to counter these defenses. A wide variety of viruses target dsRNA-induced signaling proteins, including IPS-1, interferon response factors (IRFs), interferons and interferon receptors, JAK/STAT proteins, and eIF-2α [19]–[20]. Some viral products attempt to sequester dsRNA (e.g., poxvirus E3L [21]) or to directly interfere with cellular dsRNA binding domains (e.g., HIV TAR RNA [19]–[20]). Virtually all viruses that inhibit apoptosis do so by targeting early steps in the pathway, for example by inhibiting p53, mimicking anti-apoptotic Bcl-2, or interfering with death receptor signaling [22]–[23]. Among the few viral proteins that directly inhibit one or more caspases are African swine fever virus A224L (which inhibits caspase 3) [24], poxvirus CrmA (which inhibits caspases 1, 8, and 10 but not others) [25], and baculovirus p35 (which inhibits several caspases but is relatively ineffective against caspase 9) [25].

Because PKR activation and caspase activation function in similar ways and involve proteins that have separate domains with well-defined functions, these two processes can be combined to circumvent most viral blockades [26]–[27]. In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA. If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.

For delivery into cells in vitro or in vivo, DRACOs can be fused with proven protein transduction tags, including a sequence from the HIV TAT protein [28], the related protein transduction domain 4 (PTD) [29], and polyarginine (ARG) [30]. These tags have been shown to carry large cargo molecules into both the cytoplasm and the nucleus of all cell types in vitro and in vivo, even across the blood-brain barrier.Results and Discussion Top

We produced DRACOs with different dsRNA detection domains, apoptosis induction domains, and transduction tags (Figure 1). The dsRNA detection domains included PKR1–181, PKR1–181 with dsRBM 1 (NTE3L), dsRBM 2 (CTE3L), or dsRBM 1 and 2 (2×E3L) replaced by the dsRNA binding motif from poxvirus E3L, and RNaseL1–335 (which binds to 2–5A produced by endogenous cellular 2–5A synthetases in response to viral dsRNA). The apoptosis induction domains included FADD1–90 Death Effector Domain (DED, which binds to procaspase 8), Apaf-11–97 caspase recruitment domain (CARD, which binds to procaspase 9), and murine Apaf-11–97 (mApaf) CARD. Except for mApaf, all domains refer to the human sequence. Isolated dsRNA detection domains and apoptosis induction domains were produced as negative controls. Mutant DRACOs with deleterious K64E [9] and homologous K154E mutations in the PKR domain were also produced as negative controls. Proteins were produced with TAT, PTD, or ARG tags on the N terminus, C terminus, or both termini. Proteins were expressed in BL21(DE3)pLysS Rosetta E. coli. An empty expression vector was transformed into the E. coli and the same purification protocol was followed, resulting in control extract without DRACOs.

Figure 1. A variety of DRACOs and controls were produced.

(A) DRACOs with different dsRNA detection and apoptosis induction domains were designed and produced. All domains were human except murine Apaf-1 (mApaf-1), and some dsRNA detection domains used PKR1–181 with vaccinia E3L dsRNA binding motif replacing PKR dsRBM 1 (NTE3L), dsRBM 2 (CTE3L), or both (2×E3L). His denotes His6 purification tag and Txd denotes PTD, TAT, or ARG transduction tag. DRACOs with transduction tags on the N-, C-, or both termini were produced. (B) This protein gel shows examples of DRACOs and negative controls that were produced. 1 µg was loaded per lane. Final yields were approximately 30 mg purified protein per liter of culture.doi:10.1371/journal.pone.0022572.g001

DRACO rapidly entered cells, persisted within cells for days, and mediated apoptosis in cells transfected with dsRNA. PKR-Apaf DRACO with PTD or TAT tags entered cells efficiently, whereas DRACO without a transduction tag did not (Figure 2A). DRACO entered cells within 10 minutes, reached a maximum after approximately 1.5 hours (Figures 2B, S1), and persisted inside cells for at least 8 days (Figure 2C). L929 cells transfected with both DRACO and poly(I):poly(C) dsRNA exhibited greatly increased apoptosis within 24 hours, whereas cells that received only DRACO did not (Figure 3). Pan-caspase and caspase-9 inhibitors eliminated DRACO-mediated apoptosis in the presence of dsRNA.

Figure 2. DRACOs penetrated cells and persisted for days.

(A) DRACOs with PTD or TAT tags entered H1-HeLa cells more readily than DRACO without a transduction tag. 400 nM PKR-Apaf DRACO was added to medium for 1 hour, and then cells were trypsinized and washed to remove any DRACO on the cell surface. Cells were lysed and analyzed for DRACO by westerns using anti-His6 antibodies. Lysate from approximately 105 cells was loaded in each lane. A known amount of purified PKR-Apaf DRACO was used as a standard as indicated. (B) DRACOs entered HeLa cells within 10 minutes and reached a maximum after 1.5 hours. 400 nM TAT-PKR-Apaf DRACO was added to medium for the specified time, and then cells were analyzed as in (A). (C) DRACOs persisted within HeLa cells for at least 8 days. 500 nM PTD-PKR-Apaf DRACO was added to cell medium for 1 hour, and then cells were put into DRACO-free medium. After the specified number of days, cells were analyzed as in (A).doi:10.1371/journal.pone.0022572.g002

Figure 3. DRACOs mediated apoptosis in cells containing dsRNA.

L929 cells transfected with both DRACO and poly(I):poly(C) dsRNA exhibited apoptosis within 24 hours, whereas cells that received only DRACO did not. Caspase inhibitors eliminated DRACO-mediated apoptosis in the presence of dsRNA.doi:10.1371/journal.pone.0022572.g003

We measured the viability of normal human lung fibroblast (NHLF) cells that had been treated with PKR-Apaf DRACOs or negative controls and then challenged with 130 plaque forming units (pfu) per well rhinovirus 1B (Figures 4, S2, S3). Untreated cell populations succumbed to virus within days, indicating that any innate cellular responses were ineffective against the virus or blocked by the virus. DRACOs with PTD, TAT, and ARG tags prevented significant cytopathic effects (CPE) in virus-challenged cell populations by rapidly killing any initially infected cells, thereby terminating the infection in its earliest stages. DRACOs had no apparent toxicity in unchallenged cells. Isolated PKR1–181 and Apaf-11–97 domains were nontoxic but not antiviral, even when added simultaneously (but not covalently linked). DRACO with deleterious amino acid changes also had little efficacy. Likewise, an amount of purified bacterial extract (without DRACOs) approximately 10-fold greater than the average volume of DRACOs added to cells was nontoxic and not efficacious, demonstrating that any remaining bacterial contaminants such as lipopolysaccharide did not affect the cells or produce antiviral activity. Thus the antiviral efficacy appears to require intact functional DRACOs. Tests using DRACOs with protein transduction tags on the N terminus, C terminus, or both termini indicated that N-terminal tags generally worked the best (data not shown). DRACOs with transduction tags penetrated cells and were antiviral when administered by themselves (Figures 2, S2A), but efficacy was enhanced by co-administration with Roche FuGene 6 to maximize uptake (Figure S2B), so FuGene was used in experiments unless otherwise noted. Cell viability measured 7 days post infection (dpi) showed little difference if DRACO-containing medium was removed 3 dpi after untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells after DRACOs were withdrawn (Figure 4B).

Figure 4. DRACOs were effective against rhinovirus 1B in NHLF cells.

(A) 100 nM DRACO was effective against 130 pfu/well rhinovirus, whereas 100 nM negative controls were not (12 dpi). (B) Cell viability measured 7 dpi showed little difference if 100 nM DRACO-containing medium was removed 3 dpi when untreated cells had widespread CPE from 130 pfu/well rhinovirus 1B; there was no relapse of viral CPE in treated cells after DRACOs were withdrawn. (C) 1 dose of 25 nM PTD-PKR-Apaf DRACO was effective against rhinovirus 1B in NHLF cells when it was added from 6 days before infection to 3 days after infection. (Complete viral CPE in untreated cell populations required 3–4 days in our experiments, and for these experiments a significant fraction of cells were still uninfected 3 dpi.) Cell viability was measured 14 dpi.doi:10.1371/journal.pone.0022572.g004

DRACOs were added approximately 24 hours before virus unless otherwise noted, but other dosing times were tested (Figure 4C). One dose of PTD-PKR-Apaf DRACO was efficacious against rhinovirus 1B in NHLF cells when added up to 6 days before infection, supporting the western data (Figure 2C) that DRACO persisted inside cells for at least 8 days. Up to 3 days after infection, one DRACO dose could still rescue a significant percentage of the cell population. After 3 days, virtually all of the cells had already been killed or at least infected by the virus.

Additional DRACO designs exhibited efficacy against rhinovirus (Figure 5A). Other effective dsRNA detection domains included NTE3L, CTE3L, 2×E3L, and RNaseL1–335. Other effective apoptotic domains included FADD1–90, mApaf11–97, and procaspases [26]–[27]. Although the initial performance of these alternate DRACOs was generally inferior to that of PKR-Apaf human DRACO in these experiments, better performance might be achieved with further optimization. These results demonstrate that the alternate DRACO designs are nontoxic and efficacious against virus, and they support the DRACO mechanism of action.

Figure 5. DRACOs were effective against rhinovirus 1B and other viruses.

The median effective concentration for DRACOs with PTD, TAT, and ARG tags against a variety of viruses was 2–3 nM, as illustrated for PTD-PKR-Apaf DRACO against rhinovirus 1B, murine encephalomyelitis, and murine adenovirus (Figures 5C).

DRACOs were effective against a broad spectrum of other viruses in a variety of cell types (Tables 1–2). DRACOs were effective against rhinoviruses 2 and 30 in NHLF cells (data not shown) and rhinovirus 14 in HeLa cells (Figure S4). DRACOs were effective against murine adenovirus in L929 cells if added before or up to at least 72 hours after virus (Figures 6, S5), demonstrating efficacy against a DNA virus (Figures 6A, S5), in murine cells (using human apoptotic DRACO domains to recruit endogenous murine procaspases), when treatment is delayed until significantly after infection (Figure 6B), and with a variety of DRACO designs (Figure 6C). DRACOs were effective against murine encephalomyelitis in L929 cells regardless of whether the DRACO-containing medium was removed 3 dpi (Figure 7A), whether DRACOs were added before or after infection (Figure 7B), and which DRACOs were used (Figures 7C, S6). DRACOs were effective in Vero E6 cells against Amapari and Tacaribe, arenaviruses that are closely related to lymphocytic choriomeningitis virus (LCMV), Lassa, and Junin viruses (Figures 8A, S7, S8). Likewise, DRACOs were effective against Guama strain Be An 277 (Figures 8B, S9); comparable results were obtained for Guama strain Be Ar 12590 (data not shown). Guama virus is a significant human pathogen and is closely related to other bunyaviruses such as Rift Valley fever, hantavirus, and Crimean-Congo virus. DRACOs were similarly effective against dengue type 2 (New Guinea C) hemorrhagic fever virus, a major human pathogen that is very closely related to other flaviviruses such as West Nile virus, Yellow fever virus, and Omsk virus (Figures 8C, S10, S11). DRACOs were also effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes (Figure S12 left), reovirus 3 in BALB/3T3 murine cells (Figure S12 center), and adenovirus 5 in AD293 cells (Figure S12 right).

Based on these encouraging initial animal trials, future work should be done to test and optimize antiviral efficacy, pharmacokinetics, and absence of toxicity in vitro and in vivo. Future experiments can further characterize and optimize dsRNA binding, apoptosis induction, cellular transduction, and other DRACO properties. More extensive trials are also needed to determine how long after infection DRACOs can be used successfully, or if DRACOs are useful against chronic viral infections without producing unacceptable levels of cell death in vivo.

DRACOs should be effective against numerous clinical and NIAID priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered. We have demonstrated that DRACOs are effective against viruses with DNA, dsRNA, positive-sense ssRNA, and negative-sense ssRNA genomes; enveloped and non-enveloped viruses; viruses that replicate in the cytoplasm and viruses that replicate in the nucleus; human, bat, and rodent viruses; and viruses that use a variety of cellular receptors (Table 1).Materials and Methods TopEthics statement for mouse trials

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on Animal Care of MIT (Assurance Number: A-3125-01). Guidelines to minimize suffering were followed, and avertin anesthetic was used for intranasal procedures.Cloning

Contact-inhibited cells were grown to 50–80% confluence and non-contact inhibited cells to 20–50% confluence in 96-well plates with 100 µl/well medium. DRACOs or controls were added to columns of wells, 8 wells/column. Except in Figs. 2 and S2, 0.4–1% (vol/vol) Roche FuGene 6 was co-administered with DRACOs and controls to optimize cellular uptake. Wells received virus approximately 24 hours after DRACO unless otherwise noted. On selected days, cell viability in each plate was measured using CellTiter 96 (Promega). Assay schedules, viral doses, and other parameters were optimized for different cell/virus systems. Micrographs were taken in 24-well plates under similar conditions.DRACO cell penetration assays

Cells in 24-well plates were incubated with DRACOs for varying lengths of time, then trypsinized, washed thoroughly in PBS, and lysed. Lysate from approximately 105 cells was loaded in each lane. DRACOs were detected via westerns using mouse anti-His6 (Invitrogen) and goat anti-mouse IgG HRP (Jackson).Apoptosis assays

CellTiter 96 cell viabilities were normalized to 100% for untreated uninfected and 0% for untreated virus-killed cells. Graphs indicate averages (n = 8) with s.e.m. Experiments were repeated at least 3 times with similar results.Mouse trials

7-week-old female BALB/c mice (Charles River) received DRACO i.n. (~0.5 mg in 50 µl) or i.p. (0.8–2.5 mg in 200 µl). Mice were challenged i.n. with 0.3–1.3 LD50 influenza H1N1 A/PR/8/34. Mice received DRACO i.p. once daily on days -1 and 1–3 and twice on day 0, or just one i.n. DRACO dose simultaneously with virus. Lungs were harvested on day 2 and viral titers determined by serial dilutions onto 96-well MDCK plates. For pharmacokinetics, organs were harvested at designated times, then sonicated into 1 ml PBS with 1% Triton X-100. 1 mg organ solution was mixed with 2× Laemmeli buffer, boiled 5 min., and run on a 10–20% SDS PAGE gel with a standard curve of purified DRACO, followed by western blots with anti-Apaf (Millipore) and HEP-labeled anti-rabbit IgG (Jackson Immunoresearch). Blots were developed with Pierce luminescent reagent and exposed to film. DRACO bands were quantitated by Gel Doc densitometry vs. the standards.Supporting Information Top

Figure S1.

DRACOs entered normal human lung fibroblasts. NHLF cells were incubated overnight with 500 nM PTD-PKR-Apaf DRACO labeled with Lumio (Invitrogen), washed with Hank's balanced salt solution, and photographed with a fluorescent microscope to compare (A) untreated and (B) DRACO-treated cells. DRACOs appeared to be distributed throughout each cell in both the cytoplasm and the nucleus.